During mitosis, chromosomes are accurately segregated to daughter cells by a microtubule-based structure called the mitotic spindle. It has long been recognized that undetected microtubule damage causes mitotic defects and genetic instability which renders cells susceptible to tumorigenesis. A well-established pathway, the spindle assembly checkpoint pathway, ensures equal separation of sister chromatids and is conserved in all eukaryotes. We recently uncovered evidence for a new pathway, unique to vertebrates, which can also sense microtubule stress. Combined genetic, cellular and biochemical analyses have led to the finding that CUL7 and CUL9, two cytoplasmically localized E3 ubiquitin ligases which can bind to p53, control mitosis and cytokinesis by sensing microtubule stress. Deletion of Cul9 in mice resulted in wide spread polyploidy, spontaneous tumor development, and rendered mice susceptible to carcinogenesis indicating that Cul9 is a tumor suppressor. Conversely, gain of function in CUL9 promoted a p53-dependent apoptosis. We further found that depletion of CUL7 or its binding partner OBSL1 caused severe microtubule damage, abnormal chromatid alignment, and defects in cytokinesis and widespread mitotic cell death, all of which can be rescued by simultaneous depletion of CUL9. These extensive preliminary results led us to propose that there exists in vertebrates an OBSL1-CUL7-CUL9-p53 pathway that senses microtubule stress during mitosis and is functionally separate from the well-established spindle-assembly checkpoint. We propose here a series of rigorous genetic and biochemical experiments to examine the role of this novel OBSL1-CUL7-CUL9-p53 pathway in maintaining genome integrity. In the first Aim, we will generate Cul7-Cul9 and Obsl1-Cul9 double knockout mice to genetically test the functional relationship of CUL7 and CUL9 and between OBSL1 and CUL9 in vivo. In the second Aim, we will genetically test the contribution of p53 to pathway by generating two separate knock-in mice, each with a point mutation in the p53 binding domain of CUL7 and CUL9. Finally, we will biochemically test how ubiquitylation contributes to the mechanism of the OBSL1-CUL7-CUL9-p53 pathway. PUBLIC HEALTH RELEVANCE: Many errors in mitosis lead to failure of cytokinesis and generation of tetraploidy and polyploidy which have deleterious consequence to cell proliferation and can also contribute to tumorigenesis. It has long been noted that loss of p53 function causes severe mitotic defects and development of tetraploidy. An emerging area of p53 research has suggested a potential nuclear/transcriptional-independent function of p53 in the cytoplasm. The function and molecular mechanisms of p53 in mitotic control and in cytoplasm both remain elusive. We found that CUL7 and CUL9, two cytoplasmic localized E3 ubiquitin ligases that bind to p53, constitute an OBSL1-CUL7-CUL9-p53 pathway that functions to maintain microtubule and genome integrity, likely through a regulation p53 cytoplasmic function. A better understanding of both the functions and mechanisms of the OBSL1-CUL7-CUL9-p53 pathway, a goal of this investigation, will identify novel targets for the treatment of cancer by enhancing the efficacy of anti-mitotic therapeutic agents.